Biotech and Nanotech. Torrey DeLuca HORIBA, Ltd. All rights reserved.
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1 Biotech and Nanotech Torrey DeLuca
2 Nanotechnology and Biotech Micelles Liposome Proteins Gold Nano particles DLS Zeta Potential
3 Proteins and Size Immunglobin G (antibody) Hemoglobin Insulin Andeylate kinase (enzyme) Glutamine syntetase (enzyme)
4 Gold Nanoparticles Surface Plasmon Resonance Gustav Mie, Ann. Physik25, 377 (1908)
5 Gold Nano-Particles Useful Biomarker Color Changes with Size Polymers and Proteins easily bind to gold Gold is chemically inert
6 Dilute Gold Colloid Varian Sample Used in protein screening Mie studied gold colloids Gold Colloid
7 Gold Colloids SEM (above) and TEM (below) images for RM 8011
8 Nano-technology and Horiba Research of materials with dimensions from 1 to 100 nanometers(nm) = Nanoscience LA-950: 30nm 3 mm LB nm- 1 µm DT nm 300 µm
9 Damascus sabre Multiwalled tubes Scale bars: 5 nm (a) and 10 nm (b) In b, the tubes are bent like a rope. a b Materials: Carbon nanotubes in an ancient Damascus sabre Nature 444, 286(16 November 2006)
10 Dynamic Light Scattering QELS Quasi Elastic Light Scattering PCS Photon Correlation Spectroscopy Light Scattering Incident momochromatic light Light Scattered from moving particles Wavelength shifted scattered light measured at a stationary detector Particle Size is calculated from the information contained in the fluctuating scattered light signal
11 Benefits of DLS Rapid Sensitive to aggregates R 6 scattering dependence Non-invasive Quantitative
12 Proper Measurement Large Particles or Dust The presence of a few large particles or Dust can cause the scattering intensity to fluctuate significantly These fluctuations can make measurements unusable In order to overcome these problems Introduce sample into the bottom of the cuvett to avoid washing dust off the cuvett walls Do not vortex the partially filled cuvett. Do not wash disposable cuvetts Filtration. The easiest way to remove large impurities from solution is by filtration. Centrifugation is another effective way to remove large impurities from the
13 Methods to Measure Size There are many methods that can be used to measure size or aggregation state, including Sedimentation equilibrium Size exclusion chromatography Native gel electrophoresis Light scattering Light scattering Easiest to implement, the Quickest to perform, and the Least destructive to the sample
14 Dynamic Light Scattering: Brownian motion 1 nm 1 µm Particles in suspension undergo Brownian motion due to solvent molecule bombardment in random thermal motion. Brownian Motion Random Related to Size Related to viscosity Related to temperature
15 Brownian Motion
16 LB550 Why should one consider Dynamic Light Scattering? Non-invasive measurement Can Measure Low quantities of material Can Measure Concentrated Samples Good for detecting trace amounts of aggregate Good technique for macro-molecular sizing
17 Cost of Materials Must characterize using small quantities DLS useful here Final product cost drives analysis tool
18 Diffusion Particle is randomly diffusing Larger particles will diffuse more slowly Larger particles have more Inertia Scatter light off this diffusing particle Measure the Frequency Shift of the signal Laser Detector Frequency Shifted Signal
19 Dynamic Light Scattering Measured frequency-intensity distribution (power spectrum) Power spectrum takes form of Lorentz distribution, whose half-value width can be expressed as 2Dq² All parameters in the half-width are known or measured The Diffusion Coefficient D is related to the Particle Size Frequency Shifted Signal Frequency-Intensity Distribution Iterative Calculation Stokes-Einstein
20 DLS Spectrum Analyzer Spectrum Analyzer Operates in the frequency domain of scattered light Auto Correlation Function Operates in the time domain of scattered light
21 Spectrum Analyzer Trajectory of a particle in phase space Variation of the particles position with time Spectrum analysis of fluctuating variable
22 Hydrodynamic Radius Shape Information Particles with shape Diffuse More slowly Over estimation of size
23 LB550 The range of instrument 1nm to 6μm Temperature setting up to 70 o C Concentration range up to 40wt% Low volume cuvettes 30μL Viscometer attachment
24 Range of Sizes Two particles 1nm and 1μm Volume of the 1nm particle is 1nm 3 Volume of the 1μm particle is 1,000,000,000nm 3
25 Mixed Samples You need 1 Billion 1nm particles to equal the scattering from One 1μm particle! DLS is useful for detecting these aggregates Electron Microscopy would miss these aggregates: AFM, TEM, SEM, etc
26 You get the idea So Light Scattering is an excellent technique for uncovering that single large outlier in a distribution!!! I m over here!
27 The difference between 1nm and 1μm in scale is the same as the difference between a mosquito and an elephant
28 Don t Believe Me? African elephants weigh on average 3000kg An unfed Mosquito weighs g A Well fed Mosquito can weigh 0.003g There is a 1 billion times difference in size The same difference between 1μm and 1nm
29 Say we don t care about the aggregates We want to know the size of our smallest particles That is like saying we want to know the size our mosquitoes in a herd of elephants Even if we only care about the smallest particles, can we use DLS? What happens?
30 Filter to monitor aggregation A filter will remove our aggregates Filters available in sizes 20nm to 2μm We can also centrifuge the sample and extract the supernatant
31 Particle Diameter (μm) Settling and DLS Movement due to Brownian Motion >> > > ~ < << 55.4 The Natural limit for Dynamic Light Scattering: Gravitational Settling Gravitational Settling occurs at about 1μm Movement due to Gravitational Settling
32 Filtration in Action Filtered Aggregated insulin with 20nm filter Temperature ramp up to 60 o C Aggregated Insulin Filtered Insulin - monomeric Unstable Insulin High Temperature
33 How does Concentration Affect Analysis Some ways Diffusion Drag Measured Alcholoic Emulsion LB550 Multiple Scattering Concentration limit of technique Aggregation Equilibrium Concentration limit of material Filtration has no affects
34 Bulk Viscosity Change Particles appear to diffuse together Apparent Increase in particle size No Change in distribution width Dilute Concentrated Intensity Size Diffusion Drag
35 Data Mexican Mudslide Milk Emulsion Alcoholic Beverage off the Shelf at the Grocery Store Well understood sample 200nm size with a high zeta potential at ph 7 Extremely stable sample
36 Diffusion Drag LB550 Data Use bulk viscosity for Concentrated sample Apparent size shift upwards with concentration Polydespersity- distribution width is constant Size Intensity Distribution Overlay Frequency % Mudslide neat Viscosity Corrected Mudslide dilute Mudslide neat Size (nm)
37 Tabular Data Filename New Visc Sample Name Mudslide neat Corrected Viscosity Mudslide dilute Mudslide neat Viscosity (mpa s) Median (nm) Mean (nm) CV Polydespersity Index Diffusion Coefficient (m2/s) E-15 (m2/s) E-14 (m2/s) E-14 (m2/s) Adjust viscosity parameter No change in distribution width Apparent change in size is viscosity dependent
38 Multiple Scattering Incident Light Scatters off of more than one particle Particles appear smaller in size Distribution is wider than dilute analysis Dilute Intensity Concentrated Size
39 Aggregation Equilibrium Intensity Aggregate Size You cannot filter out aggregates in concentrated state Equilibrium has been reached Filtration will cause aggregates to reform Point where equilibrium occurs is important in understanding formulation stability
40 Small Molecule Applications Protein Crystallization Protein Denaturation Protein Formulation Protein Folding Enzyme-Substrate reactions Macro-Molecular temperature melts Estimated Molecular Weight Lipid Micelle formation- CMC Macro-Molecular Characterization
41 Starburst Polymers: Dendrimers Dendrimer Mean Intensity PDI Dendrimers are repeatedly branched chain polymeric molecules AVG Expected Dendrimer Values 256 surface groups MW Size 58 kda 7.2nm
42 Size Data for Dendrimer
43 Protein Aggregation Time Study Unstabilized 10mg/ml lysozyme at ph 2 Lisa Cole and Ben Burnett at the Florida Institute of Technology
44 Stability influenced by: Temperature Protein concentration ph Ionic strength Aggregation influenced by: Freezing Exposure to air Interactions with metal surfaces Time Evolution
45 Small Molecules Antibody characterization Dynamic light scattering for molecular weight determination Protein formulation stability Quaternary Structure of Protein
46 BSA- well characterized protein DLS Can be used to determine the aggregation state of the protein 2mg/mL filtered BSA
47 DLS a Crystalliztion Monitor Size variations as a function solution properties protein concentration ph precipitant concentration temperature Monomers assemble Crystals Precipitates DLS quantifies the aggregates state Early predictions about the crystallization outcome
48 Protein Crystallization Screening Empirical correlation between DLS distribution breadth and successful crystallization When PDI polydispersity index > 0.500, only 8% chance of crystal growth When PDI is less than 0.200, then 70% chance of crystals
49 DLS and Crystallization With DLS at the center of the protein screening process the chances of growing crystals is optimized Gloria E.O. Borgstahl "How to Use of Dynamic Light Scattering to Improve the Likelihood of Growing Macromolecular Crystals"
50 Estimating Molecular Weight Empirical Models Some models take into account shape factors Deviations from Expected values indicate aggregation and dimerization
51 Drug Delivery Applications
52 Bind Biosciences* Targeting ligand provides recognition, enabling targeted nanoparticles to identify and bind to their intended target site. Surface functionalization shields targeted nanoparticles from the immune system. Polymer matrix encapsulates payload molecules in a matrix of biodegradable polymers. Therapeutic payloads include small molecules, peptides, proteins, etc. * Cambridge, MA, recent LA-950 customer
53 hates water Self Assembly: Micelles Hydrophobic tail Hydrophilic head loves water -c-h-c-h-c-h- R +/- non polar polar
54 Biotech Applications Micelles- self-assembly colloidal aggregated surfactant molecules DLS can characterize CMC CMC- Critical Micelle Concentration Point at which surfactants emulsify to form micelles
55 CMC Values
56 Triton-x100 measured CMC CMC value for Triton-x100 Measured using the LB550 The expected value is 0.021% CMC Concentration Intensity Size Triton x-100 wt% (nm) 10mMol NaCl drop drops drops drops
57 Liposomes 100 nm
58 Liposomes - Doxil
59 Liposomes Applications Lipid bilayer vesicles Sub-micron Encapsulates API (active pharmaceutical ingredients) Used in creams, emulsions and drug delivery
60 Liposomes to target tumor growth Size is critical to how the liposome Encapsulates protein Functions within body Remains stable over time Delivers the protein Novartis Liposome Data
61 Liposome and Microfluidizer After One Pass through a Microfluidics fluidizer
62 Liposome Fluidization Before fluidization After fluidization decrease in size
63 Long Term Liposome Stabilization Change in Stability as a function of ph ph adjusted to induce agglomeration Liposome sensitive to ionic and salt environment Liposome Mean (nm) PDI 1 Pass 1 Pass 1 Pass 1 Pass Before Before Before Before 1 Pass ph 12 1 Pass ph
64 Particle Stability
65 Zeta Potential and Stability An Electric Double Layer forms spontaneously around charged particles in an ionic matrix The more Diffusely the counter charge is distributed around the particle the stronger the chemical potential
66 ZETA POTENTIAL If a particle is negatively charged, a thin layer of positive charge forms around the particle (the Stern Layer). Beyond the Stern Layer, is the DIFFUSE Layer where there is a wider layer of mostly opposite charge. The potential at the surface of the particle is designated the NERNST Potential, and the potential at the outside of the Stern layer is designated the ZETA Potential. ZETA Potential is a useful because it quantifies the surface activity of colloidal particles Diffuse Layer Particle electric potential (millivolts) - Stern Layer + - REPRESENTATION OF ATTRACTED LAYERS AND ZETA POTENTIAL + Zeta Potential Nernst Potential Distance (Angstroms)
67 Zeta Potential and Stability The Diffuse Layer contains only a small fraction of counter charge 10% But, it extends far into the solution Therefore, it is of prime relevance for interactions
68 Van der Waals or London Forces Short range Attractive forces Strong force inversely proportional to size Drives Aggregation
69 The Strength of Electrostatic Interactions If all the electrons were removed from one-tenth of a cubic millimeter from the nose cone of the space-shuttle and placed on the pad The attraction would be so great between the positive charge on the cone and negative charge on the pad The shuttle would remain locked in place despite full thrust of the rockets
70 Acoustic Spectroscopy Advantages: Can accommodate high sample concentrations Can measure Zeta Potential in native concentration Signal output Detector Signal source
71 Electroacoustics Zeta Potential Piezo crystal Electrodes A Zeta Potential Probe
72 Acoustic Spectroscopy Benefits Dilution will disrupt the Zeta Potential Acoustic Spectroscopy the sample is not diluted Electro-acoustics measures Zeta Potential without dilution Non-invasive no voltage is applied to the sample- no deleterious current
73 Titration Curve BSA BSA ph Titration Zeta Potential ph
74 BSA at ph 5.6 and 4.2 BSA Mean ph Conformational changes in BSA BSA iso electric point is 5.5 There is a change in size near this point Aggregation Unfolding Conformation changes
75 Overlay dimer and aggregate Change in Size as ph is Adjusted Near the IEP (dimerized) BSA at ph extremes (aggregated)
76 Tabulated Size Data Mean CV PDI (nm) BSA ph BSA ph BSA ph BSA ph BSA ph BSA ph BSA ph BSA ph BSA ph BSA ph
77 Review DLS Quick Robust Quantitative Non-invasive Zeta Potential Formulation Stability Information Information on charge distribution Completely Non-invasive
78 Webinar Library
79 Questions? The End
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